THIS INVENTION relates to gene silencing. More particularly the invention relates to use of double-stranded RNA hairpin duplexes in gene silencing.
Short hairpin RNAs (shRNAs) expressed from RNA Pol II and Pol III promoters can be used to silence target genes. shRNAs are expressed in the nucleus, and like microRNAs (miRNAs), are shuttled to the cytoplasm where the RNA interference (RNAi) machinery produces small interfering RNA (siRNAs). Typically, shRNAs include double stranded RNA (dsRNA) duplexes of up to 30 base pairs (bp) or nucleotides, which limits the number of functional siRNAs that can be generated from the hairpin precursor.
Long ds RNA hairpins (IhRNAs) and dsRNA duplexes can also be processed by the RNAi machinery into functional siRNAs. Typically, IhRNAs and dsRNA duplexes contain at least three DICER cleavage events and are therefore about 60 bp to 66 bp or longer.
Importantly, IhRNAs are capable of generating different siRNAs, and the number of different targeted siRNAs that are generated is directly proportional to the length of the dsRNA duplex (i.e. the IhRNA). Apart from targeting a greater genetic sequence, multiple siRNAs simultaneously target different sites, preventing the possible generation of mutant variants (i.e. for viral and cancer gene sequences) which may “escape” the targeted effects of siRNAs. Mutational escape is a phenomenon observed frequently for studies using shRNAs and siRNAs against viral targets and is not only limited to the target sequence but also to flanking sequences which affect local RNA secondary structures.
There are difficulties in cloning IdsRNA hairpins, however. Additionally, long dsRNA duplexes that are used in mammalian cells are known to activate the non-specific interferon response system in mammalian cells (e.g. PKR and Rnase L pathways). These difficulties in cloning and the immuno-stimulatory effect have limited the wide-spread application of long hairpins for gene silencing in mammalian and other cells. The major difficulties with long directly inverted repeats are the formation of cruciform junctions which cause genetic instability. This could lead to the rearrangement or splicing of the DNA constructs as the cell's recombination machinery can recognize cruciforms as Holliday junctions which are substrates for homologous recombination.